Zheng Jian-Yong, Lan Xing, Li Xiao-Jun, Huang Li-Juan, Zhang Yin-Jun, Wang Zhao
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, Zhejiang, China.
School of Medicine and Life Sciences, Xinyu University, Xinyu, Jiangxi, 338004, China.
Protein Expr Purif. 2019 Mar;155:1-7. doi: 10.1016/j.pep.2018.10.012. Epub 2018 Oct 31.
Pichia pastoris expression is a mature and efficient eukaryotic expression system. In this work, Aspergillus oryzae lipase (AOL, with the molecular mass of 28 kDa), which can perform highly stereoselective hydrolysis of (R, S)-methyl 2-(4-hydroxyphenoxy) propanoate, was expressed in P. pastoris X-33. The specific activity of AOL was 432 U/mg, which was obtained by fed-batch cultivation in a 5 L bioreactor using a methanol feeding strategy. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut-off membrane and purified with DEAE-Sepharose™ FF ion-exchange chromatography and phenyl Seflnose™ 6 FF hydrophobic interaction chromatography. The purified lipase activity reached 5509 U/mg. AOL showed high activity toward short-chain triacylglyceride (C), and the optimum temperature and pH were 40 °C and 8.0, respectively. The purified enzyme activity was inhibited by Zn and Cu. Moreover, the K and V values were 1 mM and 32.89 mmol/min, respectively.
毕赤酵母表达是一种成熟且高效的真核表达系统。在本研究中，能够对（R，S）-2-（4-羟基苯氧基）丙酸甲酯进行高度立体选择性水解的米曲霉脂肪酶（AOL，分子量为28 kDa）在毕赤酵母X-33中得到表达。AOL的比活性为432 U/mg，这是通过在5 L生物反应器中采用甲醇补料策略进行分批补料培养获得的。发酵后，上清液用截留分子量为10 kDa的超滤膜浓缩，并用DEAE-Sepharose™ FF离子交换色谱和苯基Seflnose™ 6 FF疏水相互作用色谱进行纯化。纯化后的脂肪酶活性达到了 5509 U/mg。AOL对短链甘油三酯（C）表现出高活性，其最适温度和pH分别为40°C和8.0。纯化后的酶活性受到锌和铜的抑制。此外，K和V值分别为1 mM和32.89 mmol/min。
